Main focus of my work is the design and the installation of a new laser laboratory. Key point will be the experimental setup of a CARS (Coherent Anti-Stokes Raman Scattering) microscope, means the combination of a ps-CARS laser-system and a confocal microscope. This experimental technique provides a non-invasive microscopic insight into biological samples as well as samples of material-scientific interest. As a complementary method, the CARS microscopy offers many interesting properties: a label-free sample investigation due to the chemical contrast based on the vibrational Raman spectra, strong anti-Stokes Raman signals due to the coherent excitation scheme, short acquisition times, and a high spatial resolution due to the optical non-linearity of the CARS process.
The Coherent Anti-Stokes Raman Scattering (CARS) process is a four-wave mixing process based on an optical excitation with two laser energies called pump and Stokes. The excitation scheme is shown in figure 1. The difference of both laser energies is tuned to match the energy of a Raman-active vibrational mode of the relevant molecule. The high-energy Anti-Stokes signal arises due to inelastic scattering of the pump wave with the coherent excited state prepared by the interplay of pump and Stokes beam.
A scheme of the experimental setup for a dual-CARS microscope is depicted in figure 2. In this scheme three excitation pulses are generated by a Nd:Vanadate pump laser and two OPOs (optical parametric oscillator), overlapped in time and space, and coupled into a laser scanning microscope. An objective focuses the beams in the sample, and the emitted CARS signal is collected by an aspherical lens. A short-pass beam splitter separates the dual-CARS signal into its two components for detection by separate photomultiplier tubes. This experimental setup has the advantage of detecting two independent Anti-Stokes signals simultaneously. This can be used to either image two chemical species at the same time or to increase the signal-to-noise ratio of the images by comparing the signals in resonance with the Raman mode and off-resonant.
The ps-CARS laser setup in our laboratory exists of a picoTRAIN (IC-1064-15000/532-8000 ps Nd:VAN) from High-Q-Laser optically pumping two Levante Emerald OPO from APE Berlin. The arrangement of the laser and the two oscillators on the optical table in our laser lab can be seen in figure 3. The picoTRAIN can deliver pulses on the order of 6ps (76Mhz repetition rate). There are two simultaneous outputs with wavelengths of 532nm and 1064nm, and the output power can be as high as 8W and 15W, respectively.The optical parametric oscillators are synchronously pumped ps-OPO's with a signal resonant cavity and a collinear, type-I noncritical phase-matching. The output signal and idler wavelengths can be tuned between 680nm and 980nm and between 1150nm and 2300nm, respectively. This is done by the temperature change of the LBO crystal, the rotation of the birefringent Lyot-filter and the piezo-driven change of the cavity length. Combining the output of the pump laser and the two oscillators we can use picosecond pulses with up to six different wavelengths at the same time.
CARS images can be taken in forward (F-CARS) or in backward (E-CARS, epi) direction. The image below shows a typical F-CARS measurement (Cheng et al., Biophys. J. 83, 502 (2002)). NIH3T3 cells are seen in a late stage of apoptosis (cell death). The imaging contrast is based on the CH2 symmetric vibration at 2845 cm–1.